采用分子动力学模拟探究VWF-A1突变体G561S的亲和力变化机制

发布时间：2018-11-22 12:09

【摘要】：血管性血友病因子VWF(von Willebrand factor,VWF)的A1结构域与血小板膜上的糖蛋白受体GPIbα(platelet glycoprotein Ibα,GPIbα)的结合能够诱发血小板黏附和聚集以及止血栓子的形成。研究表明发生于VWF或者GPIbα分子内的突变会改变VWF与GPIbα相互作用的亲和力,从而引起血管性血友病(von Willebrand disease,VWD)。目前,VWF与GPIbα间的相互作用机制尚未完全理解。然而众多的A1晶体结构(野生型或突变体,结合状态或未结合状态)之间构象差异较小的事实为进一步解释这些突变体与其配体间的亲和力的差异造成了困难。Matthew Auton等人关于A1-GPIbα亲和力高低与A1结构域热稳定性相关的研究表明似乎不能只用静态结构上的观察和分析来研究蛋白相互作用的亲和力调控机制。之前,我们曾研究过局部动力学驱动的功能增强型A1结构域的亲和力调控机制。本研究则引入了A1的功能减弱型突变体G561S,以便探明G561S是否也服从同样的亲和力调节机制。本文采用可以观察蛋白原子具体运动细节的分子动力学模拟方法,通过分析比较2M型突变体G561S-A1(功能减弱型)、野生型WT-A1和2B型突变体R543Q-A1(功能增强型)的结构变化和动力学性质,以探究G561S突变导致2M型VWD疾病的分子结构基础,本研究可以为相关的抗血栓药物和止血药物的开发提供指导。本文首先构建了VWF-A1、G561S-A1、R543Q-A1三个A1结构的全原子模型。然后,利用MD模拟方法考察三者在生理盐水中的构象的改变,柔性的变化以及氢键/盐桥的形成与演化过程。最后,利用分别表征A1结构域柔性和稳定性的RMSF与RMSD值,并结合表征A1结构域动力学性质的其他参数,诸如螺旋间夹角,氢键/盐桥生存率等。解析野生型和突变体A1结构域与各自配体的结合亲和力差异,以便增进我们对临床上观察到的止血性缺陷的理解。我们的分子动力学模拟结果表明功能减弱型突变G561S并没有显著改变A1结构域的总体构象,突变信号没有直接传递到G561S与其配体GPIbα的结合面上。2M型突变亲和力的变化主要受到局部构象以及局部动力学性质的调节。功能减弱型突变通过降低其α2螺旋钟摆式运动幅度的方式调节其内部疏水核心的暴露,从而间接的降低其与配体的亲和力。更加详细的分析表明α2螺旋动力学性质的降低进一步受到突变位点所在的对临床上观察到的2M型止血性缺陷的理解。β2-β3 loop构象变化的影响,本文表明那些能够调控α2螺旋以及N末端的运动状态的路径,对于抗血栓药物和止血药物的开发至关重要。本文的研究结果也有利于增进我们
[Abstract]:The binding of the A1 domain of von Willebrand factor VWF (von Willebrand factor,VWF to GPIb 伪 (platelet glycoprotein Ib 伪, GPIb 伪, a glycoprotein receptor on platelet membrane, can induce platelet adhesion and aggregation and the formation of hemostatic emboli. Studies have shown that mutations occurring within VWF or GPIb 伪 may alter the affinity of VWF to GPIb 伪 interaction, leading to (von Willebrand disease,VWD). At present, the mechanism of interaction between VWF and GPIb 伪 has not been fully understood. However, a large number of A1 crystal structures (wild type or mutant, The fact that the conformational differences between the binding and unbound states) are small, further explain the differences in the affinity between these mutants and their ligands, resulting in difficulties for. Matthew Auton et al. In relation to the level of A1-GPIb 伪 affinity and the A1 domain Studies related to thermal stability suggest that it is not possible to study the affinity regulation mechanism of protein interactions only by static structural observation and analysis. Previously, we have studied the affinity regulation mechanism of the functionally enhanced A1 domain driven by local dynamics. In order to find out whether G561S is also accepted by the same affinity regulation mechanism, the attenuated mutant G561S of A1 was introduced in this study. In this paper, a molecular dynamics simulation method, which can observe the specific motion details of protein atoms, is used to analyze and compare the G561S-A1 (functional attenuated type) of the 2m mutant. The structural changes and kinetic properties of wild-type WT-A1 and type 2B mutant R543Q-A1 (functional enhanced type) were studied to explore the molecular structural basis of G561S mutation leading to 2m type VWD disease. This study may provide guidance for the development of antithrombotic drugs and hemostatic drugs. In this paper, a full atomic model of three A1 structures of VWF-A1,G561S-A1,R543Q-A1 is constructed. Then, the conformation, flexibility and formation and evolution of hydrogen bond / salt bridge were investigated by MD simulation. Finally, the values of RMSF and RMSD were used to characterize the flexibility and stability of A1 domain, respectively, and other parameters, such as helical angle, hydrogen bond / salt bridge survival rate, were combined to characterize the dynamic properties of A1 domain. The differences of binding affinity between wild type and mutant A1 domain and their ligands were analyzed in order to improve our understanding of clinically observed hemostatic defects. Our molecular dynamics simulation results show that the reduced function mutation G561S does not significantly change the overall conformation of A1 domain. The mutation signal was not transmitted directly to the binding surface between G561S and its ligand GPIb 伪. The variation of the affinity of the 2m mutation was mainly regulated by the local conformation and the local kinetic properties. The functionally attenuated mutation regulates the internal hydrophobic core exposure by reducing the amplitude of 伪 2 helical pendulum motion, thus indirectly reducing its affinity to the ligand. More detailed analysis showed that the reduction of 伪 2 helical kinetic properties was further influenced by the understanding of the mutation site to the clinically observed 2 M hemostatic defect. The conformation of 尾 2- 尾 3 loop was affected by the change in the conformation of 尾 2- 尾 3. It has been shown that the pathways that regulate the movement of 伪 2 helix and N terminal are essential for the development of antithrombotic and hemostatic drugs. The results of this paper are also beneficial to us.
【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R543

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